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provided by Electronic Publication Information Center The ISME Journal (2010), 1–9 & 2010 International Society for Microbial Ecology All rights reserved 1751-7362/10 $32.00 www.nature.com/ismej ORIGINAL ARTICLE Carbon and nitrogen fluxes associated with the cyanobacterium Aphanizomenon sp. in the Baltic Sea
Helle Ploug1,3, Niculina Musat2,3, Birgit Adam2, Christina L Moraru2, Gaute Lavik2, Tomas Vagner2, Birgitta Bergman1 and Marcel MM Kuypers2 1Department of Botany, Stockholm University, Stockholm, Sweden and 2Department of Nutrient Group, Max Planck Institute for Marine Microbiology, Bremen, Germany
Carbon and nitrogen fluxes in Aphanizomenon sp. colonies in the Baltic Sea were measured using a combination of microsensors, stable isotopes, mass spectrometry, and nanoscale secondary ion mass spectrometry (nanoSIMS). Cell numbers varied between 956 and 33 000 in colonies ranging in volume between 1.4 10 4 and 230 10 4 mm 3. The high cell content and their productivity resulted in steep O2 gradients at the colony–water interface as measured with an O2 microsensor. Colonies were highly autotrophic communities with few heterotrophic bacteria attached to the filaments. 3 1 Volumetric gross photosynthesis in colonies was 78 nmol O2 mm h . Net photosynthesis was 3 1 3 1 64 nmol O2 mm h , and dark respiration was on average 15 nmol O2 mm h or 16% of gross photosynthesis. These volumetric photosynthesis rates belong to the highest measured in aquatic systems. The average cell-specific net carbon-fixation rate was 38 and 40 fmol C cell 1 h 1 measured by microsensors and by using stable isotopes in combination with mass spectrometry and nanoSIMS, respectively. In light, the net C:N fixation ratio of individual cells was 7.3±3.4. Transfer of
fixed N2 from heterocysts to vegetative cells was fast, but up to 35% of the gross N2 fixation in light was released as ammonium into the surrounding water. Calculations based on a daily cycle showed
a net C:N fixation ratio of 5.3. Only 16% of the bulk N2 fixation in dark was detected in þ Aphanizomenon sp. Hence, other organisms appeared to dominate N2 fixation and NH4 release during darkness. The ISME Journal advance online publication, 29 April 2010; doi:10.1038/ismej.2010.53 Subject Category: microbial ecosystems impacts Keywords: Aphanizomenon; nitrogen fixation; ammonium release; photosynthesis; respiration
Introduction represented by unicellular picocyanobacteria (Stal et al., 1999; Stal and Walsby, 2000). In the absence of Aphanizomenon sp. is a filamentous, colony- other nitrogen sources, these large filamentous forming N2-fixing cyanobacterium, which frequently cyanobacteria cover their nitrogen demand through blooms in lakes and brackish waters. It occurs both N2 fixation, and growth is probably limited by the as colonies and single trichomes during their availability of phosphate (Moisander et al., 2003; growth, which often lasts 2–3 months during the Walve and Larsson, 2007). Aphanizomenon sp. summer in the Baltic Sea (Rolff et al., 2007; accumulates inorganic phosphate during the growth Degerholm et al., 2008). The primary productivity season, and blooms appear to terminate when the in the Baltic Sea by filamentous cyanobacteria, internal storage is used up and bulk concentrations which are represented by Aphanizomenon sp., are low in mid-August in the Baltic Sea (Walve and Anabaena sp., and Nodularia spumigena, represents Larsson, 2007). 44% of the community primary production. These N2 fixation by filamentous cyanobacteria, includ- filamentous organisms comprise 20–30% of the ing Aphanizomenon sp., is considered to be a cyanobacterial biomass in the Baltic Sea. The quantitatively important source of nitrogen to the remaining primary productivity and biomass is plankton community during the summer months in the Baltic Sea (Degerholm et al., 2008). Nitrogen budgets of the Baltic Sea have suggested that Correspondence: H Ploug, Department of Botany, Stockholm Aphanizomenon leaks a substantial fraction of its University, Lilla Frescativa¨gen 5, Stockholm SE-10691, Sweden. fixed nitrogen that is used by other members E-mail: [email protected] 3These two authors contributed equally to this work. (for example picocyanobacteria and heterotrophic Received 21 December 2009; revised 13 March 2010; accepted 15 bacteria) in the plankton community (Larsson March 2010 et al., 2001; Stal et al., 2003; Rolff et al., 2007). C- and N-fluxes in Aphanizomenon sp. H Ploug et al 2 Using stable isotopes, it was shown that 5–10% of transferred to an Eppendorf vial with 1.5 ml sur- 15 the N2 fixed by large cyanobacteria (Aphanizomenon rounding water, and fixed with a drop of 5% Lugol’s sp. and Nodularia sp.) in the Baltic Sea is found in solution. The fixed colonies were stored cold at 4 1C smaller organisms represented by the 2–5 mm size until further analysis. Colonies disintegrate in Lugol’s class, for example picocyanobacteria, after 8–10 h solution, and the filaments sediment at the tip of the incubation time (Ohlendieck et al., 2000). Direct Eppendorf vial; 1 ml containing all filaments from measurements of ammonium or dissovled organic each colony was pipetted into a gridded Sedgewick nitrogen (DON) release relative to N2-fixation rates, Rafter counting chamber (Wildlife supply Company). however, have not been performed in field-sampled Numbers and dimensions of dispersed filaments, cyanobacteria from the Baltic Sea. vegetative cells, and heterocyst frequency (in no Nanoscale secondary ion mass spectrometry filament per length, and in percent of vegetative (nanoSIMS) is a novel technique, which reveals cells) in each colony was measured under an inverted elemental and isotopic surface composition after microscope (Zeiss, Switzerland) equipped with a measurement of isotopic composition at a spatial digital camera (Axio Cam, Zeiss) connected to an resolution of 50 nm. Combining this technique with image analysis program (AxioVision, Zeiss). Total isotope enriched incubations has enabled measure- cumulative filament length per field was measured ments of N2 and C fixation on a single-cell level in until the mean value was stable and the s.d. was mixed populations (Musat et al., 2008). Hence, o2% of the mean value. using this technique we can now ascribe N2 and C Total abundance of Aphanizomenon sp. trichomes fixation to individual cells of different organisms in and cells in bulk water and in concentrated samples the Baltic Sea. To understand small-scale carbon (see below) was measured using the same counting and nitrogen fluxes in Aphanizomenon sp. colonies, procedure as described above. Five replicates, each we combined microsensors and stable isotope comprising a subsample of 1 ml of 50 ml sample enrichment incubations with mass spectrometry as fixed in Lugol were analyzed both for in situ bulk well as with nanoscale secondary ion mass spectro- water as well as concentrated samples. metry (nanoSIMS). Using microsensors, gross and net photosynthesis as well as dark respiration was
measured directly in colonies as a function of Small-scale measurements of O2 fluxes colony size and cell content in field-sampled Freshly sampled colonies were transferred to a Petri Aphanizomenon sp. in the Baltic Sea. Using stable dish coated by a 3 mm thick agar layer (1% w:w) at isotope in combination with mass spectrometry and the bottom and covered by filtered (0.2 mm) water nanoSIMS, we measured carbon and nitrogen fixed from the sampling site. The diffusion coefficients of by Aphanizomenon sp. cells and its release to the gases, ions, and solutes in 1% agar are close to those surrounding water. in (sea) water (Libicki et al., 1988; Revsbech, 1989). Artifacts from a solid boundary, which limits solute exchange between colonies and the surrounding Materials and methods water, was, therefore, avoided by using a Petri dish coated with agar (Ploug and Grossart, 1999). The Sampling Petri dish was placed in a thermostated container at In August 2008, Aphanizomenon colonies were in situ temperature. Oxygen concentrations were sampled in the upper 10 m of the water column at measured at the colony–water interface using a 0 0 station B1 (N 581 48 28, E 171 37 60) in the Clark type oxygen microsensor (Revsbech, 1989) Stockholm archipelago using a plankton net (Hydro- attached to a micromanipulator. The current was bios: 0.5 m diameter, mesh size: 90 mm). Water at 5 m measured by a picoamperemeter (Unisense PA 2000) depth was collected by a water sampler. The salinity connected to a strip-chart recorder (Kipp and was 6 and the temperature was 19 1C. Samples were Zonen). The electrode was calibrated at anoxic immediately brought to the laboratory in which they conditions and at air saturation. Its 90% response were analyzed by microsensors or incubated with time was o1 s and the stirring sensitivity o0.3%. stable isotopes. The oxygen microelectrode tip was 2 mm wide and its position was observed under a dissection micro- scope with an ocular micrometer. The electrode was Microscopy manually approached toward the colony until it All three dimensions of individual colonies were visually touched the surface. Oxygen-concentration measured under a dissection microscope using a gradients were measured at the colony–water inter- calibrated ocular micrometer. Their volume was face at 50 mm step increments. Three replicates of calculated as ellipsoids: Vol ¼ 4/3p a b c, the O2-concentration gradient were measured in where a, b, and c are the half-axes in the three light and in dark, respectively. The light source was dimensions. A microsensor was used to lift and turn a Schott lamp (KL 1500 LCD) equipped with an colonies on an agar plate covered with water from infra-red cutoff filter and calibrated using an LiCOR the sampling site. Each colony with known size was scalar irradiance sensor. The fluxes of oxygen were thereafter collected using a wide bore pipette, measured at steady state of concentration gradients
The ISME Journal C- and N-fluxes in Aphanizomenon sp. H Ploug et al 3 at the colony–water interface, and calculated from coupled to a Delta Plus Advantage mass spectro- Fick’s first law of diffusion: meter was used (Montoya et al., 1996). Before analysis, GF/F filters were freeze dried overnight dC and dehydrated in an HCl-smoke environment in a J ¼ D ð1Þ desiccator overnight. Round punchouts (Ø 1 cm) of dr the GF/F filters were packed into tin cups and were tightly pressed into a pill-shaped form. These were 2 1 where J is the flux (nmol O2 cm s ), D is the then loaded onto an autosampler flushed with diffusion coefficient of O2 in the bulk water helium, which automatically dropped the packed 5 2 1 (2.02 10 cm s at 6 and 19 1C; Broecker and filters into the combustion furnace. As a standard, Peng, 1974), and dC/dr is the radial-concentration caffeine was used for calibration. 3 gradient of O2 (nmol O2 cm ). Surface area and volume was calculated as for ellipsoids (Mass, 1994). Carbon production rates were calculated NanoSIMS analysis assuming a photosynthetic quotient of 1.2 mol O2: For NanoSIMS analysis of single Aphanizomenon 1molCO2. cells, Gold-Palladium-coated GTTP filters contain- ing chemically fixed water samples were cut with a round stencil (Ø 5 mm) and mounted on a sample Incubations with stable isotopes holder. The analysis was performed using a Nano- Water samples containing 1.10 108 Aphanizo- SIMS 50 l manufactured by Cameca (Gennevilliers, menon sp. cells per liter (±0.03 108 cells l 1; s.e.) France). For each individual cell, we recorded were incubated in 250 ml serum bottles by adding simultaneously secondary ion images of 12C, 13C, colonies harvested by the plankton net to bulk water 12C14N, and 12C15N using four electron multipliers. samples. Microscopy revealed that the mean The measurements and the image and data proces- trichome size was 230±95 mm, and most heterocysts sing were performed as described earlier (Musat were observed within the trichomes. Hence, cells et al., 2008). did not appear damaged by the sampling procedure. The 15N/14N and the 13C/12C ratios of individual The serum bottles were closed with rubber stoppers Aphanizomenon cells were determined for experi- through which 13C bicarbonate was injected to a ments with and without (that is control experi- 15 14 final bicarbonate concentration of 1.83 mM (11% ments) labeled substrate. Subsequently, the N/ N labeling) except for three control bottles. Using a gas and 13C/12C enrichment of individual cells was 15 mouse, 2 ml N2 (Sigma, Germany) was injected to calculated by subtracting the average cellular ratios the sample (12% final labeling). The syringe was of Aphanizomenon in the control experiments from flushed several times with Argon gas between the ratios obtained for individual cells from the injections. All bottles were thoroughly shaken labeling experiment. during 5 min to ensure an even distribution of 15 dissolved N2 in the samples. Many colonies remained intact, and microscopy did not reveal cell Ammonium analysis 15 þ damage during this procedure. Three replicates were The net release of NH4 from cyanobacteria was incubated for 0, 3, or 6 h in light at 300 mmol quantified in the filtrate after the incubations with 15 photons m 2 s 1, as measured using an LiCor scalar N2 during 0, 3, and 6 h in light or darkness using irradiance sensor, or in dark at in situ temperature the method by Warembourg (1993). Samples were (19 1C) in a thermostated room. The incubations analyzed on a GC-IR mass spectrometer (VG Isogas were stopped by filtration of samples onto Limited, Middlewitch, UK). þ pre-combusted GF/F filters (for EA-MS analysis) The NH4 microenvironment of colonies was (Montoya et al., 1996). The filtrates were filled into modeled, knowing the diffusive properties of colo- 12 ml gas tight exetainers and killed by adding nies from O2 measurements, the net carbon fluxes, 100 ml saturated HgCl solution to each exetainer. the C:N fixation ratio, and fraction of fixed nitrogen 2 þ Subsamples for nanoSIMS analysis were fixed with released as NH4 . We used an analytical diffusion 2% paraformaldehyde and washed before filtration model by Ploug et al. (1997), with a diffusion þ 5 2 1 onto Gold-Palladium-coated GTTP filters (pore size, coefficient of NH4 of 1.6 10 cm s at 6 and 0.22 mm; diameter 25 mm; Millipore, Germany). 19 1C (Li and Gregory, 1974).
Results EA-IRMS analysis To determine the amount of 15N gas and 13C The number of cells in the Aphanizomenon sp. bicarbonate incorporated into biomass, water sam- colonies was a significant function of colony volume ples were filtered onto glass fiber filters and (Figure 1). The largest colonies had a volume of 3 analyzed through mass spectrometry using N2 and 0.023 mm with an equivalent spherical diameter CO2 released by flash combustion in excess oxygen of 340 mm, but most colonies were considerably at 1050 1C. An automated elemental analyzer smaller. The number of cells in individual colonies
The ISME Journal C- and N-fluxes in Aphanizomenon sp. H Ploug et al 4 35000 250
m) 250
30000 µ 200 )
25000 -1 200 -1 20000 150
15000 150 100 Cells colony 10000
50 (mm filaments colony Cumulative filament length 100 5000
0 0 50 0.000 0.005 0.010 0.015 0.020 0.025 Colony volume (mm3) 0
Figure 1 Number of cells and cumulative filament length (both Radial distance from colony surface ( shown as symbols) in Aphanizomenon colonies as a function of colony volume. The line represents linear regression between the 270280 290 300 310 320 330 340 variables (please see text). µ O2 ( M)
Figure 2 O2 gradients measured at the colony–water interface during dark (closed symbols) and in light at 300 mmol photons varied from 956 to 33 000 for colonies ranging 2 1 4 4 3 m s (open symbols). Each symbol represents the average value between 1.4 10 and 230 10 mm in volume. of three series of measurements with the s.d. of the mean value
The average number of cells per colony was shown as bars. The curve represents the O2-concentration 6 described as Cells ¼ 1.53 10 (Volcol), where col- gradient fitted to the measured values and used for flux ony volume is measured in mm3. Alternatively, calculations. biomass of filamentous cyanobacteria is measured as meter per length (m l 1) (Hajdu et al., 2007; Rolff et al., 2007; Degerholm et al., 2008). The cumulative 1.4
length of filaments in individual colonies varied )
-1 1.2 between 7 and 246 mm, and it was described by L ¼ 11 341 (Volcol); where length, L, is expressed in 1.0 mm. The significance level was R2 ¼ 0.61; n ¼ 37;
colony h 0.8 Po0.0001 for both expressions. The mean cell 2 3 volume was 132 mm , and the mean volume fraction 0.6 occupied by cells in colonies was 0.19. Heterocysts were 1.5% of total cells or 2.02±0.95 mm per 0.4 filament independent of colony volume. 0.2 The cell content and their productivity in colonies 0.0 gave rise to significant oxygen-concentration gradi- exchange (nmol O 2 ents at the colony–water interface (Figure 2). Oxy- -0.2
gen concentrations at the colony surface varied up to Net O 65 mM between the light (300 mmol photons m 2 s 1) -0.4 and the dark exposed colonies (Figure 2). The gradients were measurable up to 200 mm from the 0.0000.005 0.010 0.015 0.020 surface of a 340 mm large colony. Net photosynthesis Colony volume (mm3) 1 1 was 1.1 nmol colony h , and dark respiration was Figure 3 Net photosynthesis rate (open symbols) and dark 1 1 0.3 nmol colony h as calculated from the oxygen respiration rate (closed symbols) measured as a function of colony gradients measured in light and dark, respectively. volume. Each symbol represents the average value of three series of Hence, dark respiration was 22% of gross photo- measurements with the s.d. of the mean value shown as bars. synthesis in this example. The net photosynthesis and dark respiration measured as a function of colony size are shown respectively, independent of colony size. Combining (Figure 3). Net photosynthesis as well as dark these correlations with the biomass distribution respiration increased proportionally to colony (Figure 1), and assuming a cell-specific carbon volume. Net photosynthesis was described by content of 0.11 pg C mm 3 (Helcom, 1988) yielded a 2 NP ¼ 64 (Volcol); R ¼ 0.92 (n ¼ 14; Po0.001), and carbon-specific net photosynthesis rate of 1 1 dark respiration was described by R ¼ 15 (Volcol); 0.029 h and a dark respiration rate of 0.007 h R2 ¼ 0.94 (n ¼ 14; Po0.001), where volume is mea- independent of colony size. Average cell-specific sured as mm3, and NP and R measured as photosynthesis and respiration rates were 42 and 3 1 1 1 1 1 nmol O2 mm h . Thus, volume-specific gross 10 fmol O2 cell h or 38 and 8.3 fmol C cell h , photosynthesis, net photosynthesis, and dark respectively, assuming a photosynthetic quotient of 3 1 respiration was 79, 64, and 15 nmol O2 mm h , 1.2 (Table 1).
The ISME Journal C- and N-fluxes in Aphanizomenon sp. H Ploug et al 5 Table 1 Net photosynthesis and dark respiration in Aphanizomenon sp.
Method Net photosynthesis Dark respiration
nmol C m 1 h 1 fmol C cell 1 h 1 nmol C m 1 h 1 fmol C cell 1 h 1
Microsensora 5.4 38 1.3 8.3 NanoSIMS 40±12 — — aAssuming a photosynthetic quotient of 1.2.
Figure 4 Distribution of 12C14N (a, d, g), and 15N/14N(e, h), and 13C/12C(f, i) in single Aphanizomenon cells as measured by nanoSIMS. a–c: no label, d–f: t ¼ 3h, g–i: t ¼ 6 h. The size bars are 2 mm.
NanoSIMS analysis of colonies and filaments percent labeling are shown in the bulk in Figure 5. incubated with stable isotopes of C and N showed These varied largely between single cells after 6 h. a rapid transfer of 15N from heterocysts to vegetative Less C relative to N was incorporated into hetero- cells. An example is shown in Figure 4. 13C carbon cysts as compared with vegetative cells. In vege- was evenly distributed in vegetative cells after 3 h tative cells, the average cell-specific carbon assimilation incubation time with only low assimilation into rate was 40±12 fmol C cell 1 h 1, and the average heterocysts. Heterocysts were also less enriched in cell-specific N assimilation into biomass was 15N as compared with vegetative cells. The occur- 4.8±3.2 fmol N cell 1 h 1 in light. Hence, the average rence of N-storage products, for example cyano- net C:N assimilation ratio into individual vegetative phycin, was detectable after 3 h as regions with cells was 7.3±3.4 in light (Table 2). Dark respiration 15 1 1 locally intense labeling of N (in cyanophycin was 8.3 fmol C cell h and dark net N2-fixation granules). Hence, incorporation of 15N into proteins rate was 0.28±0.15 fmol N cell 1 h 1. of heterocysts seemed low, and most of the 15N label We tracked the release of fixed 15N by measuring 15 þ was transported to adjacent vegetative cells. the production of NH4 in the surrounding water 13 15 þ Cell-specific assimilation rates of C and N (Table 2). The release of NH4 was 35% of the total derived from nanoSIMS analysis and converted to gross N2 fixation in light. The corresponding value total net C and total net N assimilated by using was 89% in darkness. The C:N ratio of gross
The ISME Journal C- and N-fluxes in Aphanizomenon sp. H Ploug et al 6 production (gross photosynthesis:gross N2 fixation) were similar to those reported in cultured þ was, therefore, 6.3 (Table 2). The NH4 microenvir- colonies of Aphanizomenon sp. (Carlton and Pearl, onment of colonies was modeled, knowing the 1989). Volume-specific gross photosynthesis was 3 1 diffusive properties of colonies from O2 measure- 79 nmol O2 mm h independent of colony size. ments, the net carbon fluxes, the C:N fixation ratio, Hence, there was no indication of diffusion limita- þ and fraction of fixed nitrogen released as NH4 . The tion of photosynthesis by CO2 or bicarbonate in þ NH4 -concentration gradients in a colony with O2 these colonies. Volume-specific gross photosynth- gradients similar to the colony shown in Figure 2 are esis was 10- to 80-fold higher than those measured þ shown in Figure 6. The NH4 concentration at the in surface scums dominated by Nodularia sp. in the colony surface was 5.5 mM when the bulk concentra- Baltic Sea (Ploug, 2008) and of other cyanobacterial tion was 0.15 mM. Hence, ammonium was 437-fold surface blooms including Aphanizomenon in inland enriched inside colonies as compared with the bulk lakes (Ibelings and Maberly, 1998). This high rate is water concentration. among the highest reported in aquatic systems, comparable with or higher than those measured in shallow sediments and microbial mats (Jørgensen Discussion et al., 1983; Villbrant et al., 1990; Lassen et al., 1992). This study is the first to combine microsensors with Carbon-specific net photosynthesis rates were mass spectrometry and nanoSIMS techniques to similar to optimal rates reported using the 14C understand small-scale fluxes of carbon and nitro- method in field-sampled Aphanizomenon from the gen in a Baltic Sea cyanobacterium among those Baltic Sea (Walve and Larsson, 2007) as well as in dominating the summer blooms. It was directly cultures of Aphanizomenon isolated from the Baltic shown that Aphanizomenon is highly productive Sea in the same area as that of this study (Degerholm even during the late stage of its bloom and season in et al., 2006). The 14C method provides an estimate of August. High concentrations of biomass in colonies photosynthesis, which is usually considered to and their productivity lead to significant O2 gradi- represent a rate between gross and net photosynth- ents within these colonies and their surrounding esis (that is the net carbon retainment in the cell). water. The O2 gradients measured during light Using microsensors, it was possible to determine net ) -1 70 h -1 60
50
40
30
20
10
Total C assimilation rate (fmol cell 0 0 2 4 6 8 10 12 Total N assimilation rate (fmol N cell-1 h-1)
Figure 5 Cell-specific carbon and N2-fixation rates as measured Figure 6 Ammonium gradient at colony–water interface of by nanoSIMS. Vegetative cells are shown as open symbols, and Aphanizomenon colony modeled from measured O2 fluxes, þ heterocysts as closed symbols. C-, and N2-fixation rates, and NH4 -release rates.
+ 15 Table 2 Gross and net C:N fixation ratio, N2-fixation rate, and net NH4-release rate in Aphanizomenon sp. traced by N